FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] Propane 1, 3-diol hydrochloride), Mediates Cytotoxicity through Caspase Independent and Protein Phosphatase 2A Dependent Mechanisms in Chronic Lymphocytic Leukemia and Lymphoblastic Leukemia/Lymphoma.

FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] propane 1, 3-diol hydrochloride) is a synthetic compound produced by modification of a natural immunosuppressant, ISP-1. It is an immunosuppressive agent that is being developed to prevent organ transplant rejection. Recent studies indicate additional role for FTY720 in inducing cell apoptosis. We demonstrate here a novel mechanism by which FTY720 mediates cytotoxic effects in cell lines representing different B cell malignancies and primary B cells from chronic lymphocytic leukemia (CLL) patients. FTY720 induced apoptosis as detected by annexin V/ propidium iodide staining in representative B cell lines and CLL patient derived CD19⁺ B cells in time and dose dependent manner (p<0.0001, untreated vs 10mM-treated CLL cells, n=15). In contrast to previous reports in T cell lines, FTY720 induced cytotoxicity in Raji cell line and primary CLL cells is independent of activation of caspase 3, 8 and 9 or poly-ADP ribose polymerase cleavage. Further, pan-caspase inhibitor Z-VAD-fmk rescued these cells from fludarabine but not FTY720 induced apoptosis (p=0.001 fludarabine vs fludarabine+z-VAD-fmk; p=0.99 FTY720 vs FTY720+z-VAD-fmk, n=5). Over-expression of Bcl-2 failed to protect transformed B-cells from FTY720 induced apoptosis suggesting Bcl-2 independent cytotoxic effect. Interestingly, FTY720 induced consistent increase in protein phosphatase 2a (PP2a) activity and concentrations of okadaic acid that inhibited the FTY720-induced PP2A activity also resulted in inhibition of FTY720-mediated cytotoxicity in B cell lines and primary CLL cells, indicating a role for PP2A activation in FTY720 induced cytotoxicity. Consistent with its activation of PP2A, FTY720 induced dephosphorylation of of Erk1/2 in CLL B cells. Further, FTY720 treatment resulted in significant in-vivo therapeutic efficacy associated with prolonged survival in a xenograft SCID mouse model of disseminated B cell lymphoma/leukemia (median survival time for FTY720 treated mice was 47 days (95 %CI 39–53) compared to 18 days in placebo controls (95% CI 17–19) P<0.0001-FTY720 vs placebo). These results provide first evidence for a PP2A dependent and caspase independent cytotoxicity of FTY720 in B cells. The novel caspase and Bcl-2 independent mechanism of cytotoxicity concurrent with identification of PP2a activation as a surrogate marker of cell killing provide further justification for clinical development of this agent in lymphoid leukemia.